GLYCOWORKS RapiFluor-MS QUICK START PROTOCOL

Significance of the Topic

Glycan profiling plays a pivotal role in the characterization of biologic therapeutics, influencing safety, efficacy, and regulatory compliance. Rapid and reproducible glycan analysis is essential for biopharmaceutical development, quality control, and batch comparability.

Objectives and Overview

This Quick Start Protocol outlines a streamlined workflow for processing up to 24 glycoprotein samples in a 3 × 8 format. It integrates rapid deglycosylation, efficient labeling with RapiFluor-MS reagent, and HILIC-based cleanup to generate MS-ready glycan derivatives in under one hour.

• 24-sample throughput in a microplate configuration
• Minimal hands-on time
• Prepared samples directly compatible with LC-MS analysis

Methodology

The protocol is divided into three key steps:

1. Rapid Deglycosylation
   • Reconstitute the glycoprotein to 1.5 mg/mL and add 3% (w/v) RapiGest SF surfactant.
   • Heat at 90 °C for 3 minutes, cool to room temperature, add Rapid PNGase F, and incubate at 50 °C for 5 minutes.
2. Rapid Labeling of Glycosylamines
   • Dissolve 9 mg of RapiFluor-MS reagent in 110 µL anhydrous DMF.
   • Add 10 µL of labeling solution to the deglycosylation mixture, incubate 5 minutes at room temperature, then dilute with acetonitrile.
3. HILIC Cleanup of Labeled Glycans
   • Condition and equilibrate a GlycoWorks HILIC µElution Plate with water and 85% acetonitrile.
   • Load the acetonitrile-diluted sample, wash with 1% formic acid in 90% acetonitrile, elute with SPE Elution Buffer, and dilute eluate with SPE Diluent.

Instrumentation

• Heat blocks maintained at 90 °C and 50 °C
• GlycoWorks HILIC µElution Plate (microplate format) with waste and collection trays
• Rapid Buffer, RapiGest SF surfactant, Rapid PNGase F enzyme
• RapiFluor-MS reagent, anhydrous DMF, acetonitrile, SPE Elution Buffer and Diluent

Key Findings and Discussion

This protocol achieves complete deglycosylation and labeling within 15 minutes, delivering high labeling efficiency and recovery. The HILIC cleanup ensures removal of excess reagents and surfactant, producing purified glycan pools suitable for sensitive MS detection.

Benefits and Practical Applications

• Accelerated glycan sample preparation for biopharmaceutical QA/QC and research
• High reproducibility and minimal sample handling variability
• Compatibility with downstream LC-MS or CE-LS methods for detailed glycan profiling

Future Trends and Applications

Further developments may include automation with liquid-handling platforms, integration into online LC-MS workflows, and adaptation for complex or low-abundance glycoprotein targets. Emerging labeling chemistries and microfluidic cleanup devices will continue to enhance throughput and sensitivity.

Conclusion

The GlycoWorks RapiFluor-MS Quick Start Protocol provides a rapid, reliable, and high-throughput solution for N-glycan analysis, supporting robust biotherapeutic characterization and quality assurance.

References

• Waters Corporation. GlycoWorks RapiFluor-MS Quick Start Protocol, Document #720005470EN, December 2020.
• Waters Corporation. Application Note 720005506EN.
• Waters Corporation. Application Note 720007038EN.